Clostridium perfringens (“Cp”), a gram-positive anaerobe, is associated with a number of diseases including gas gangrene, sudden infant death syndrome, and necrotic enteritis in chickens (Titball et al., 1999). Necrotic enteritis (NE) is a serious, fatal, and prevalent disease in the poultry industry (Justin et al., 2002) that is characterized by acute enterotoxemia, resulting in small intestine necrosis and up to 50% mortality, primarily in chickens and turkeys from 2-12 weeks of age.
Preventive treatment with antibiotics is the predominant method currently employed to prevent Cp infections in the poultry industry. Due to widespread concern over development of antibiotic resistant bacterial strains, there is a long-felt need for an alternate method.
Clostridium perfringens encodes a gene for an exotoxin defined as α-toxin. The α-toxin of Cp (cpa) is reported to be the major virulence determinant of necrotic enteritis, inducing the debilitating necrotic lesions (Logan et al., 1991). C. perfringens type A strain is the most prevalent environmental strain and produces high levels of α-toxin (Ginter et al., 1996).
The α-toxin is a 370 amino acid (AA), zinc-dependent phospholipase C (PLC) that possesses both enzymatic and toxic properties (Justin et al., 2002). Although cpa is a phosphatidylcholine-preferring phospholipase C (PC-PLC), toxicity results from its ability to hydrolyze phosphatidylcholine and sphingomyelin phospholipid substrates, both of which are key components of eukaryotic cell membranes (Justin et al., 2002). In addition to the overt properties of hemolysis, necrosis, vascular permeabilization, and platelet aggregation, the toxin elicits a variety of subtle effects on eukaryotic cell metabolism, including activation of the arachidonic acid cascade, and stimulation of protein kinase C activity (Ginter et al., 1996).
The α-toxin consists of two domains, an α-helical N-terminal domain and an eight-stranded β-sandwich C-terminal domain. The N-terminus (AA residues 1-246) contains the phospholipase C active site and three associated zinc ion binding sites. The C-terminus (AA residues 256-370) is responsible for calcium-dependent membrane binding. Its role is to facilitate the interaction of the α-toxin with membrane phospholipids. The C-terminal domain is required for phospholipid recognition as well as for hemolytic activity of the α-toxin (Titball et al., 1999).
Fragments of the α-toxin of Clostridium perfringens have been produced and tested for independent function. In mice, antibodies that cross-reacted with the full-length α-toxin were induced after immunization with either the N-(Cpa1-249) or C-terminal (Cpa247-370) domain of the toxin. Smaller fragments of the α-toxin did not induce cross-reacting antibody. In vitro, anti-Cpa1-249 neutralized phospholipase C activity but not hemolytic activity of the toxin. Anti-Cpa247-370 neutralized both the phospholipase C and hemolytic activities. Of the N-terminal and C-terminal domain fragments, only immunization with Cpa247-370 induced protection against the lethal effects of the toxin in vivo. Additionally, immunization with Cpa247-370 provided protection in a mouse model against the whole organism, C. perfringens type A. This study confirmed the essential role of α-toxin and, specifically, the cpa C-terminal domain in the pathogenesis of gas gangrene (Williamson and Titball, 1993).
U.S. Pat. No. 5,851,827 (Titball and Williamson) relates to peptides and vaccines for inducing production of antibodies directed against Clostridium perfringens α-toxin in animals. Those peptides comprise the amino acid sequence of the alpha-toxin from amino acid 247 to 370 but lack the epitopes necessary for phospholipase C and/or sphingomyelin hydrolysing activity between amino acids 1 to 240. Further provided are antisera and antibodies raised to the peptides and vaccines and particularly monoclonal antibodies and hybridoma cell lines for their production.
The α-toxin produced by Clostridium perfringens strain NCTC 8237 was shown to differ from the α-toxins produced by most strains of C. perfringens isolated from human and calves at the following AA positions: Ala174 to Asp174; Thr177 to Ala177; Ser335 to Pro335. However, these differences did not alter the toxic properties of the protein. Further, a C-terminal domain vaccine derived from this strain was demonstrated to protect against the α-toxin from a bovine enteric strain of C. perfringens (Ginter et al., 1996).
Site-directed mutagenesis of Clostridium perfringens α-toxin has been the subject of numerous studies to elucidate amino acids essential for the toxic properties of this protein. Nagahama et al. (1995) reported a single point mutation in of AA H-68 or H-148 replaced with G (H68G or H148G) resulted in complete loss of hemolytic, phospholipase C, sphingomyelinase, and lethal activities of the toxin. However, antigenicity to wild type α-toxin antiserum was retained. The same outcome resulted from a H148L substitution. A H126G, H136G, or H136A mutation significantly decreased, but did not eliminate, the toxic activities. Mutation at H-46, -207, -212, or -241 showed no effect on the biological activities, indicating these residues are not essential for toxicity. Wild-type toxin and the variant toxins at H-68, -126, and -136 contained two zinc atoms. The variant toxin at H-148 possessed only one zinc atom, suggesting that H-148 tightly binds one zinc atom which is essential for the active site of α-toxin and that neither zinc atom associated with the wild type toxin is coordinated to H-68, -126, or -136.
Guillouard et al. (1996) based a series of site-directed mutagenesis studies on the crystal structure of a PC-PLC from Bacillus cereus, as the N-terminal domain of the Clostridium perfringens α-toxin is highly homologous to its complete phospholipase C. AA substitutions of D56N, H126S, H68S, H148S, H136S, E152S, H11S, or W1S resulted in significant reduction or complete elimination of biologic activities.
Also based on the known structure of B. cereus, Nagahama et al. (1997) investigated the role of D-56, D-130, and E-152 in hemolytic, phospholipase C (PLC), and sphingomyelinase (SMase) activities of Clostridium perfringens α-toxin. The replacement of D-56 in α-toxin with E, N, G, or S resulted in complete loss of hemolytic, PLC, and SMase activities. The variant toxins at D-130 showed an approximately 100-fold reduction of biological activities compared to that of the wild-type toxin. The substitution of G for E-152 caused complete loss of these activities and retained antigenicity to wild type α-toxin antiserum. However, E152Q or E152D resulted in significant but not complete elimination of toxicity.
Martin and Hergenrother (1998) studied the role of D-55 in general base catalysis by the PC-PLC from B. cereus, the AA position analogous to D-56 in Clostridium perfringens α-toxin. Substitutions were made with L, N, or E, with L resulting in the largest reduction in catalytic activity (9×10−5% of wild type).
Available Clostridium perfringens α-toxin (cpa) sequence is highly conserved in bovine and mammalian isolates, as determined from GenBank submissions. Many publicly available DNA sequences from chicken isolates do not extend into the C-terminal domain (GenBank Accession Numbers AAL85329, AAL85330, AAL85331, AAL85332). A full-length alpha toxin sequence from swan has been published (GenBank Accession Number AF204209) and the encoded protein is highly divergent from the alpha toxin isolated from Strain 13, a human isolate (GenBank Accession CLOPLC05) (Justin et al., 2002). Recently, full-length protein sequences of cpa from 25 chicken isolates of C. perfringens were compared (Sheedy et al., 2004) and found to have only small differences in amino acid sequence (one to six differences from the Strain 13 standard). These sequence variants were grouped into five types (I-V).